Major depressive disorder (MDD) is one of the most prevalent and debilitating illnesses world wide, affecting ~17 percent of the population and causing enormous personal and economic burden. The impact of MDD is underscored by the limitations of currently available medications, including low response rates, treatment resistant patients, and time-lag (weeks-months). These data highlight a major unmet need for more efficacious and faster-acting antidepressant agents. Recent studies demonstrate that a single low dose of ketamine, a glutamate-NMDA receptor antagonist, produces rapid antidepressant actions (2 hr) that last for up to 7 days in treatment resistant patients. This rapid action, by a mechanism completely different from typical monoamine reuptake inhibitors, represents one of the most significant advances in the field of depression over the past 6 decades. We have reported that ketamine causes a rapid increase of synaptic connections in the medial prefrontal cortex (mPFC), which targets and corrects the synaptic deficits caused by chronic stress and depression. Despite this progress, the cellular mechanisms underlying the synaptic actions of ketamine, and increased glutamate transmission have not been determined. We hypothesize that the initial trigger for the rapid actions of ketamine is blockade of NMDA receptors that stimulate tonic firing of GABA interneurons, resulting in disinhibition of glutamate transmission and increased synapse formation in mPFC. Alternatively, ketamine could act directly on pyramidal neurons. This application describes an integrated multidisciplinary approach, including molecular, biochemical, electrophysiological, morphological, and behavioral studies to test this disinhibition hypothesis. Aim 1 will use cell type specific knockdown of NMDA receptors on GABA interneuron subtypes as well as pyramidal neurons in the mPFC. Based on pharmacological evidence, the GluN2B subunit will be targeted using viral expression of floxed-GluN2B shRNA and cell specific Cre recombinase transgenic lines. Preliminary results indicate that the rapid behavioral actions of ketamine are blocked by GluN2B knockdown on GABA interneurons, consistent with the disinhibition hypothesis. Aim 2 will extend these studies by characterizing NMDA/GluN2B receptor regulation of somatostatin (SST) and parvalbumin (PV) interneuron subtypes by patch recordings in reporter mice to identify the cellular basis for differences in NMDA and ketamine sensitivity of these interneurons. The influence of ketamine on inhibitory plasticity will also be determined, and the role of GABA interneuron subtypes in the actions of ketamine will be tested using cell specific optogenetic approaches. Stress and depression are reported to alter GABA neurotransmission with greater effects in women than men. Aim 3 will use reporter mice to determine the role of GABA interneuron subtypes in the effects of chronic stress and ovarian steroids, including molecular, cellular, and transcriptional responses. Characterization of the cellular mechanisms that underlie the actions of ketamine, chronic stress, and ovarian steroids will provide novel targets for safer, rapid-acting antidepressants.